Unique among histone protein families, the histone H2A family comprises three subfamilies which have significant, evolutionarily conserved protein sequence differences among themselves. The H2A1 subfamily, which is cell cycle regulated, comprises about 80% of the H2A content of mammalian chromatin while the H2AX and H2AZ subfamilies, which are cell cycle independent, and comprise about 10% each. Understanding the function of these two H2A species may lead to new insights into function of chromatin. The following studies are in progress this year to elucidate the different roles of the H2A species. (1) We have developed procedures for expressing the three human H2A histones in bacteria to prepare sufficient quantities of H2AX and H2AZ to use in various structural and functional assays. We have found that the H2AZ-H2B dimer is unstable at physiological salt conditions compared to the H2AX-H2B and H2A1-H2B dimers. There are also differences in the nucleosome reconstituted from these three H2A species. This work is being prepared for publication. (2) We are continuing to study the specific Ca++-activated phosphorylation of H2AX in mammalian cells. Using recombinant H2AX, which is a good substrate for the enzyme, we have shown that the kinase requires the Ser- G1n motif with the Ser four residues away from the C-terminal. We have evidence that the phosphorylation is induced in vivo under conditions of stress. We have also isolated a recombinant form of the HIV protein p17 which is part of the integration complex and which has a C terminal sequence highly homologous with that of H2AX. In this light, we are examining the ability of p17 to become phosphorylated. (3) X-ray crystallographic studies of the octamer with H2AX and H2AZ replacing H2A.1 are being planned in collaboration with Prof. Moudrianakis. In collaboration with Dr. Randall Morse, we are trying to express the three human H2A species in yeast to see if any of the three can replace the yeast H2A in its chromosomes. Preliminary results suggest that none of the human H2A species can replace yeast H2A; these results are interesting since Tetrahymena H2A is able to replace yeast H2A. However, little progress has been made in these two collaborations due to charges in their laboratories.